Previous attempts to achieve mixed chimerism across the xenogeneic barrier of pig-to-primate have all failed, largely due to high levels of primate natural antibodies to the Gal antigen, expressed on the cell surface of all mature pig hematopoeitic cells. Thus, following mixed chimerism induction protocols in non-human primates, we have had evidence for long-term engraftment of pig hematopoietic stem cells (HSC), which do not appear to express Gal. However, despite antibody absorptions, inhibition of complement and various drug treatments, natural anti-Gal antibodies persisted and/or recurred, and appeared to play an important role in inhibiting the progeny of pig HSC from repopulating primate recipients. The recent availability of galactosyltransferase (Gal-T) knockout (GalT-KO) swine, derived from our most highly inbred line of miniature swine, has now made it possible to study pig-to-baboon xenotransplantation in the absence of effects of natural anti-Gal antibodies. The goal of this proposal is to utilize these new GalT-KO animals as hematopoietic cell donors for the establishment of mixed xenogeneic chimerism in the pig-to-baboon combination. Ourprevious studies and our preliminary data all suggest the feasibility of applying the mixed chimerism approach to induction of transplantation tolerance across this barrier, an approach already used successfully in this laboratory for allografts and concordant xenografts in rodents, pigs and cynomolgus monkeys. Specifically, we will: 1) Optimize the engraftment of HC from GalT-KO miniature swine in conditioned baboons; 2) Examine the role of selected innate immune and non-immune factors in promoting and/or resisting engraftment of GalT-KO HCin baboons; and 3) Test the role of GalT-KO pig HC engraftment in baboons enabling the induction of tolerance in recipients of subsequent organ xenografts. Aim 3, in particular, will apply the findings of Aims 1 and 2, as well as strategies developed in the other projects of this Program Project, to the mixed chimerism approach for inducing transplantation tolerance to organ xenografts in this preclinical, discordant species combination. In addition, the experiments planned will provide basic information on xenogeneic stem cell engrafment and on the immunologic pathways responsible for xenogeneic rejection and tolerance induction in primates. As such, these studies should have both theoretical and practical implications for the eventual application of xenotransplantation as a clinical modality.